Transient decrease of hepatic NAD+ and amino acid alterations during treatment with valproate:
new insights on drug-induced effects in vivo using targeted MS-based metabolomics
Supplementary Data for:
Transient decrease of hepatic NAD+ and amino acid alterations during treatment with valproate: new insights on drug-induced effects in vivo using targeted MS-based metabolomics
Marco F. Moedas 1,3, Arno G. van Cruchten3, Lodewijk IJlst3,
Wim Kulik3, Isabel Tavares de Almeida1,2, Luísa Diogo4,
Ronald J. A. Wanders3§ and Margarida F. B. Silva1,2§
1Research Institute for Medicines – iMed.ULisboa, Metabolism and Genetics Group, Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal
2Department of Biochemistry and Human Biology, Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal
3Laboratory Genetic Metabolic Diseases, Department of Clinical Chemistry and Pediatrics, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, the Netherlands
4Centro de Desenvolvimento Luís Borges, Hospital Pediátrico de Coimbra, Coimbra, Portugal
(§These authors should be considered as equal last authors)
Table of contents
PageSupplementary material and methods / 3
Rat liver sample preparations / 3
Amino acid and kynurenine profiling in rat liver extracts using UPLC-MS/MS / 3
Supplementary figures / 5
Figure S1 / 5
Figure S2 / 6
Figure S3 / 7
Figure S4 / 8
Figure S5 / 9
Supplementary tables / 10
Table S1 / 10
Table S2 / 11
Supplementary references / 12
Supplementary material and methods
Rat liver sample preparations
NAD(P)(H), amino acids and kynurenine intermediates were extracted from rat liver samples using an adaptation of a published protocol (Masson et al. 2010). Briefly, 15 mg of snap frozen liver (wet weight) were weighed into 2 mL tubes and lysed in an 80% methanol solution containing the respective internal standard (I.S.), either 10 nmol of acetyl-pyridine adenine dinucleotide (APAD, for NAD(P)(H) quantification) or a mix of stable isotope-labelled amino acids (for amino acids and kynurenine analysis), with concentrations in the 1 to 2 mM range. Lysis of liver tissue was performed with a Tissue Lyser (Qiagen) at 25Hz for 5 mins. The lysate was centrifuged at 16,000g for 10 mins at 4ºC. Pellets were resuspended in 0.1 M NaOH and protein quantified using the BCA assay (Thermo Scientific). The supernatant was transferred to a clean 1.5 mL tube and evaporated until dryness (Eppendorf 5301 Concentrator). Dried extracts were then resuspended in a 97:3 (water:methanol) solution for NAD(P)(H) analysis or 0.01% heptafluorobutyric acid (HFBA) for amino acid quantification. Amino acids and kynurenine intermediates quantifications were performed in a Waters Quattro Premier XE UPLC-MS/MS with reverse-phase chromatography and positive ionization mode with Multiple Reaction Monitoring (MRM) detection.
Amino acid and kynurenine profiling in rat liver extracts using UPLC-MS/MS
Quantification of amino acids and kynurenine intermediates in liver samples was achieved using a modification of published procedures (Casetta et al. 2000; Nagy et al. 2003; Pitt et al. 2002). The study of the kynurenine pathway included the following intermediates: kynurenine, kynurenic acid, anthranilic acid, 3-hydroxy-anthranilic acid, nicotinamide (NAM) and nicotinamide mononucleotide (NMN). All measurements were carried out on a Waters Quattro Premier XE UPLC-MS/MS. The MS was operated in the positive mode. Data acquisition and peak integration were performed with Masslynx 4.1 software (Waters). Detection of metabolites was performed in the multiple reaction monitoring mode (MRM). The general settings were as follows: The ESI capillary voltage was 3.0 kV, extractor voltage 3 V, cone voltage 20 V. The desolvation gas (hydrogen) flow was 900 L/h with the temperature set at 300°C, the cone gas (nitrogen) flow was 50 L/h with the source block temperature set to 120°C. Individual metabolite MRM transitions and corresponding instrument parameters were identified by direct infusion of a standard solution dissolved in 0.01% HFBA. The chromatographic separation of all compounds was achieved on a reverse phase column, AcquityTM UPLC® BEH C18 (1.7 µm, 100 x 2.1 mm). The elution started with 100% of mobile phase A (0.1% HFBA), which was kept constant for 2 minutes, followed by a linear gradient in which mobile phase B (80% Acetonitrile) was increased to 50% in a period of 5 minutes up to 100% in 1 minute. After 10 seconds, the column was re-equilibrated with 100% mobile phase A for 3 minutes. The temperature of the auto-sampler was set to 10 ºC and the separation column was kept at 50 ºC. The injection volume was 10 µL with partial loop injection with needle overfill. The flow rate was set to 0.5 mL/min.
Supplementary figures
Fig. S1 Schematic representation of the acute and sub-chronic treatment of Wistar rats with valproic acid (VPA): After one single administration of drug at two doses (groups A, B), rats were sacrificed at 1h (acute group). Another group was treated daily with the lower tested dose (group C) and sacrificed at end of two weeks (sub-chronic group) at 1h after last administration.
Fig. S2 Plasma amino acids levels significantly altered in rats under sub-chronic (15 days, n=10 [Controls] and n=12 [VPA]) or acute (1 dose, n=10) administration of VPA at two doses (100 or 500 mg/kg), as determined by GC-FID. Boxplot representation of the normalized levels is based on the ratio of individual values with the average value of the respective controls. (* p<0.05, ** p<0.01 (t-test or Mann-Whitney statistics when applicable))
Fig. S3 Resolution of NADP(H) metabolites: Chromatogram (TIC, total ion current) by UPLC-MS/MS of NAD+ and related metabolites analysis, using the settings given in table 2 and the experimental conditions indicated in text, which shows the complete baseline separation within a runtime of five minutes.
Fig. S4 Liver NAD(P)(H) levels from rats under sub-chronic chronic (15 days, n=10 [Controls] and n=12 [VPA]) or acute (1 dose, n=10) administration of VPA at two doses (100 or 500 mg/kg), where significant alterations were observed as compared with respective control animals. Boxplot representation of the normalized levels is based on the ratio of individual values and the average value of the respective controls. (*p<0.05, **p<0.01 (t-Test or Mann-Whitney statistics when applicable))
Fig. S5 Simplified scheme of NAD+ biosynthesis pathways. (A) De novo biosynthesis is initiated by tryptophan (provided by diet) and proceeds through a multiple enzyme pathway, producing intermediates of the kynurenine pathway and nicotinic acid derivatives. (B) “Salvage” biosynthesis initiates from the dietary intake of NA, NAM and NR and proceeds through common intermediates such as NaAM or NMN.
Supplementary tables
Table S1. Characterization of the effects of VPA treatment on human plasma amino acid profiles: Changes in amino acid concentrations in plasma (expressed in µmol/L) from patients under chronic treatment with VPA compared with non-treated controls.
Amino acid / Control (n= 39) / Treated (n= 33)Tryptophan / 59 ± 15 / 40 ± 13 **
Alanine / 354 ± 99 / 442 ± 134 **
Methionine / 20 ± 7 / 13 ± 6 **
Glycine / 276 ± 93 / 335 ± 89 **
Leucine / 126 ± 33 / 139 ± 44
Valine / 254 ± 56 / 253 ± 62
Histidine / 86 ± 19 / 79 ± 21
Isoleucine / 64 ± 21 / 80 ± 25 **
Lysine / 173 ± 56 / 211 ± 72 *
Phenylalanine / 50 ± 9 / 55 ± 15
Ornithine / 88 ± 31 / 104 ± 52
Serine / 148 ± 34 / 220 ± 54 **
Proline / 190 ± 70 / 273 ± 109 **
Asparagine / 45 ± 9 / 23 ± 16 **
* p<0.05 vs control; independent samples t-test
** p<0.01 vs control; independent samples t-test
Table S2. Characterization of the effects of VPA treatment on rat hepatic amino acid profiles: Amino acid concentrations in liver (expressed in µmol/mg protein) from rats subjected to single or repeated administration of VPA. Controls were accordingly administered with vehicle, as depicted in supplementary figureS1.
15 Day Regimen / 1 Dose RegimenAmino acid / Control
(n=10) / VPA
100 mg/kg (n=12) / Control
(n=10) / VPA
100 mg/kg (n=10) / VPA
500 mg/kg (n=10)
Tyrosine / 0.61 ± 0.22 / 0.55 ± 0.15 / 0.47 ±0.07 / 0.62 ± 0.08* / 0.72 ± 0.19*
Glycine / 6.2 ± 1.3 / 8.4 ± 1.7* / 5.6 ± 1.1 / 8.7 ± 1.7** / 10.5 ± 3.3**
Alanine / 19.2 ± 3.7 / 16.6 ± 3.9 / 17.2 ± 2.7 / 16.1 ± 2.9 / 8.9 ± .46
Methionine / 0.54 ± 0.06 / 0.55 ± 0.08 / 0.41 ± 0.07 / 0.55 ± 0.16 / 0.53 ± 0.16
Valine / 0.89 ± 0.15 / 0.83 ± 0.10 / 0.67 ± 0.11 / 0.89 ± 0.26 / 0.89 ± 0.3
Leucine / 1.36 ± 0.20 / 1.29 ± 0.19 / 1.04 ± 0.12 / 1.43 ± 0.48 / 1.28 ± 0.37
Isoleucine / 0.64 ± 0.09 / 0.59 ± 0.10 / 0.49 ± 0.07 / 0.69 ± 0.27 / 0.63 ± 0.19
Serine / 3.62 ± 0.50 / 4.18 ± 1.41 / 2.71 ± 0.81 / 5.12 ± 1.8* / 2.98 ± 0.76
Proline / 0.78 ± 0.28 / 0.64 ± 0.19 / 0.59 ± 0.16 / 0.59 ± 0.19 / 0.45 ± 0.14
Asparagine / 0.69 ± 0.08 / 0.69 ± 0.15 / 0.58 ± 0.14 / 0.82 ± 0.32 / 0.76 ± 0.16
Ornithine / 1.44 ± 0.34 / 1.32 ± 0.26 / 1.18 ± 0.18 / 1.36 ± 0.38 / 1.69 ± 0.48
Citrulline / 0.14 ± 0.03 / 0.14 ± 0.03 / 0.15 ± 0.04 / 0.15 ± 0.04 / 0.15 ± 0.09
Tryptophan / 0.23 ± 0.03 / 0.23 ± 0.03 / 0.19 ±0.04 / 0.23 ± 0.06 / 0.24 ± 0.05
Phenylalanine / 0.66 ± 0.12 / 0.59 ± 0.11 / 0.51 ± 0.07 / 0.64 ± 0.18 / 0.56 ± 0.17
Lysine / 2.91 ± 0.49 / 4.24 ± 0.47** / 2.29 ± 0.42 / 3.66 ± 0.43** / 4.99 ± 0.93*
* p<0.05 vs control
** p<0.01 vs control
Supplementary references
Casetta, B., Tagliacozzi, D., Shushan, B., & Federici, G. (2000). Development of a method for rapid quantitation of amino acids by liquid chromatography-tandem mass spectrometry (LC-MSMS) in plasma. Clinical Chemistry and Laboratory Medicine. doi:10.1515/CCLM.2000.057
Masson, P., Alves, A. C., Ebbels, T. M. D., Nicholson, J. K., & Want, E. J. (2010). Optimization and evaluation of metabolite extraction protocols for untargeted metabolic profiling of liver samples by UPLC-MS. Analytical Chemistry, 82(18), 7779–7786. doi:10.1021/ac101722e
Nagy, K., Takáts, Z., Pollreisz, F., Szabó, T., & Vékey, K. (2003). Direct tandem mass spectrometric analysis of amino acids in dried blood spots without chemical derivatization for neonatal screening. Rapid Communications in Mass Spectrometry : RCM, 17(9), 983–90. doi:10.1002/rcm.1000
Pitt, J. J., Eggington, M., & Kahler, S. G. (2002). Comprehensive screening of urine samples for inborn errors of metabolism by electrospray tandem mass spectrometry. Clinical Chemistry, 48(11), 1970–80.
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